The present invention is directed to antiviral purine nucleosides, purine nucleoside analogs, and prodrugs of both. It is also directed to processes for increasing the antiviral activity of the compound 3'azido-3'deoxythymidine ("AZT") by administering AZT in combination with such purine compounds.
AZT has shown promise as an antiviral agent, useful in the treatment of certain viral infections which are believed to be caused by retroviruses. In particular, AZT has shown promise against the human immunodeficiency virus.
AZT is currently in use for the treatment of AIDS in patients with AIDS related complex and for use in AIDS patients who have had recent episodes of pneumocytis carinii-related pneumonia. It appears to be effective in decreasing levels of human immunodeficiency virus ("HIV") antigen in human blood peripheral monocytes. Chaisson et al., N. Engl. J. Med., Vol. 315, p. 1610-1611 (1986).
AZT is a thymidine analog in which the 3'-hydroxy group is replaced with an azido group. On entering the cell, AZT is phosphorylated to AZT monophosphate by the same cellular kinase which phosphorylates thymidine. AZT monophostate is then phosphorylated to the diphosphate and triphosphate forms by other cellular kinases. AZT triphosphate is said to interfere with retroviral RNA dependent DNA polymerase (or "reverse transcriptase"), thus inhibiting viral replication. In vitro tests have also been said to support a mechanism whereby AZT triphosphate is incorporated by virus reverse transcriptase into DNA copies of HIV after the virus enters the cell and begins to replicate. The growing DNA chairs which incorporate AZT triphosphate are thereby permaturely terminated because AZT cannot receive an additional nucleotide at the 3' position. AZT has a somewhat greater affinity for viral reverse transcriptase than for human DNA polymerases. Thus, HIV replication can be slowed without blocking cellular replication.
The phosphorylation of AZT to AZT monophosphate by thymidine kinase has been suggested as the rate limiting step in this process. Richman and Carson, Journal of Experimental Medicine, Vol. 166, p. 1141-1149 (October, 1987). Richman and Carson stated that AZT could not be phosphorylated to the triphosphate form in the peripheral blood macrophages of normal humans in high enough concentrations to inhibit the AIDS virus reverse transcriptase. They suggested that the low level of incorporation of AZT was related to a four-fold lower level of the enzyme thymidine kinase as compared to the lymphoblast line CEM. It was reported that this lower activity of thymidine kinase was associated with a nine-fold lower accumulation of AZT nucleotides, and that the nine-fold lower level of AZT nucleotides was associated with a 100 to 1000 fold reduction in antiviral activity in macrophages as compared to CEM cells.
It is suggested here that at least two important parameters affect how much AZT triphosphate is incorporated into newly formed viral nucleic acids and/or how effectively reverse transcriptase is inhibited: (1) the amount of AZT triphosphate present and (2) the amount of thymidine triphosphate "TTP") present. It is believed that AZT triphosphate competes with TTP for incorporation into newly synthesized nucleic acids. Thus, a portion of the reduction in effectiveness of AZT reported by Richman and Carson may have been related to factors other than phosphorylation; for example, macrophages may have inherently higher TTP pools than CEM cells. TTP pools were, however, not measured. The importance of the amount of TTP present as a parameter for AZT incorporation is supported by the effect of the drug ribavirin on HIV replication in the presence of AZT. Boght et al., Science, Vol. 235, p. 1376-1379 (March, 1987), reported that ribavirin reduced the effectiveness of AZT against the AIDS virus in both peripheral blood lymphocytes and H9 cells and that ribavirin decreased phosphorylation of AZT to the mono-, di-, and triphosphate forms by approximately a factor of ten in both AIDS infected and uninfected cells. The authors proposed that ribavirin could act by diminishing AZT triphosphate formation through an increase in the TTP pool which results in a feedback inhibition of the enzyme thymidine kinase, thus reducing the phosphorylation of AZT to AZT triphosphate by that enzyme, or by reducing the interaction of AZT triphosphate with the HIV reverse transcriptase enzyme.
The use of AZT does have certain drawbacks. It is unknown, for example, whether AZT can be tolerated over a prolonged time. Current clinical experience is still limited to a certain extent and the long-term effects of chronic AZT treatment remain to be evaluated. It is known, on the other hand, that AZT has numerous side effects, including bone marrow toxicity. Thus, the most common side effect is macrocytic anemia, which may be severe enough to require blood transfusions, and which has been reported to eventuate in up to 45% of patients having a CD4 (T4) lymphocyte count of .ltoreq.200/mm.sup.3. Granulocytopenia has been reported to occur in up to 55% of patients having a CD4 count of .ltoreq.200/mm.sup.3 and in up to 40% of patients with CD4 count of &gt;200/mm.sup.3 (19% and 13% of these patients, respectively, showed granulocytopenia with placebo); Physician's Desk Reference, page 820 (42d Edition 1988). It is possible that patients with folate or vitamin B12 deficiencies may be more sensitive to the bone marrow depression caused by AZT. In many cases, patients can tolerate a reduced dose of AZT with resultant improvement in white blood cell count and anemia. Severe headache, nausea, insomnia and myalgia are also reported at a significantly greater rate in AZT recipients. Id.
AZT has also been shown to have anti-bacterial activity. There have been reports of an enhanced effect from treatment of bacteria with both AZT and the anti-folate antibiotics, namely the sulfonamides. See Elwell et al., Antimicrobial Agents and Chemotherapy, Vol. 31, p. 274 (1987). The authors did not speculate on the mechanism of the observed increased sensitivity to AZT in the presence of sulfonamides; however, they did report their clinical observations that treatment of patients having pneumocystis carini pulmonary infection with both AZT and sulfonamides appeared useful.
The enzyme S-adenosylhomocysteine ("SAH") hydrolase catalyzes the reversible hydrolosis of S-adeonylhomocysteine to adenosine and L-homocysteine. When the concentration of these substrates is higher than in the micromolar range, the reaction favors synthesis. Ueland, Pharmalogical Reviews, Vol. 34, p. 223-253 (1982). There are a number of acyclic and carbocyclic analogs of adenosine which are reported to owe their antiviral activity to inhibition of SAH hydrolase. De Clercq, Biochemical Pharmacology, Vol. 36, p. 2567-2575 (1987). No SAH hydrolase inhibitors have been reported to have anti-human retroviral or anti-HIV activity.
Pharmacologic properties of AICA riboside (5-amino-1-.beta.-D-ribofuranosyl-imidazole-4-carboxamide), including metabolism and metabolic effects, were described by Thomas et al., Journal of Cellular Physiology, Vol. 103, p. 335-344 (1981). AICA riboside was discovered in the 1950's in the culture media of bacteria grown in the presence of sulfonamide antibiotics. The antifolate properties of the sulfonamides cause a block in purine metabolism that causes a build up of AICA riboside monophosphate in the cells which is then catabolized and excreted as AICA riboside. Following studies using Chinese hamster lung fibroblasts Thomas et al. wrote that AICA riboside, at a concentration of 200 .mu.M, arrested cell growth associated with pyrimidine starvation which was reversed by addition of the pyrimidine uridine. The article also refers to a vast literature on the toxic effects of purine nucleosides such as adenosine and deoxyadenosine on cell growth. Other compounds, such as EHNA and deoxycoformycin, which inhibit adenosine deaminase and cause a build up of adenosine and deoxyadensine, are also cytotoxic through pyrimidine starvation. These other molecules have additional cytotoxic effects in addition to simple pyrimidine starvation. Studies have been done on adenosine and deoxyadenosine as cytotoxic immunosuppressive compounds. Gruber et al., Annals of the New York Academy of Science, Vol. 51, p. 315-318 (1985).
Work on AICA riboside by Thomas et al., supra, led to their conclusion of an expansion of the ATP and GTP pools combined with a depression of the phosphoribosyl-pyrophosphate (PRPP) pool at 200 .mu.M AICA riboside. The purine pool (ATP and GTP) was said to have been expanded by about 40% at 50 .mu.M and 200 .mu.M AICA riboside. At 50 .mu.M and 200 .mu.M AICA riboside, the PRPP availability, as measured by adenine incorporation, dropped by 18% and 82% respectively. It was believed that the continued effect of an increase in the purine pools and a decrease in PRPP availability resulted in an inhibition of the enzyme orotate phosphoribosyl transferase-orotate decarboxylase (OPRT-ODC). This enzyme converts oratate into uridine monophosphate. Cells grown at concentrations of 50 .mu.M and 200 .mu.M AICA riboside exhibited a reduced growth rate which was reversible by the addition of uridine to the growth media and which showed an accompanying increase in orotate pools in the growth media surrounding those cells. Thomas et al. concluded that AICA riboside caused pyrimidine starvation by its effects on the ATP, GTP and PRPP pools. The inhibitory effects reported appear to be the result solely of pyrimidine starvation, whereas other compounds which caused pyrimidine starvation had numerous other cytotoxic effects.
Thomas et al. also reported that UTP and CTP pools decreased by a factor of approximately ten. TTP pools were not measured It was reported that at a much higher dose, 700 .mu.M AICA riboside, the ATP and GTP pools were no longer elevated but there was still a reduction in the PRPP pools. At that AICA riboside concentration and with that combination of observed effects there was no cytotoxicty, no oratate excretion, but still a depletion of UTP and CTP. Because there was still normal growth, the lack of oratate may have indicated a compensated pyrimidine depletion.
While AZT is the first antiviral agent to be marketed with an indication for the treatment of AIDS, it is acknowledged to be very expensive and toxic, it requires frequent dosing, and it is available only in limited supply. Thus, it will be understood that more readily available antiviral agents having less toxicity, or agents which will lessen the side effects of AZT, or both, are desired and would be of great utility. The present invention provides such agents.